PMB Midterm 2

Knocking out genes is relatively easy to understand. What is needed to "knock-in" new DNA based on your accumulated knowledge..... the answer is..... Basically, you need to add 2 different recombinant DNAs 1) one to produce Cas9 and guide RNA, and 2) another carrying your GOI that also contains regions of homology (complementarity) on either side of the site where the Cas9 has made a cut....these latter regions will allow recombination into the chromosome. These regions of homology are cloned onto the ends of the GOI. This is shown in a more complex slide where an antibody to malaria was inserted into a genome.

"Knock-In" requires: the CRISPR Cas9 system and guide RNA the antibody gene regions of homology to allow recombination into the chromosome

Name 5 effects of "cornification" and give a sentence of explanation for each.

1) Corn Surpluses Most of our corn supply goes into animal feed or is processed into starches, corn oil, ethanol, or sweetners 2) Animal Confinement Feedlots Confinement facilities house thousands of livestock along with heavy concentrations of animal wastes, odor pollution, and a dependence on antibiotics 3) Food Deserts Most of the crops from monoculture specialization are exported and leaves the regional economy 4) The Obesity Crisis Linked to the subsidized and super-sized processed foods (such as soft drinks) 5) The Dead Zone Nutrient & chemical runoff from farms in the Corn Belt flow through watersheds that empty into the Mississippi River and create a "dead zone" in the Gulf of Mexico, harming fish and marine life

What are the 6 toxic substances used in growing potatoes?

1) Fumigant is added to soil before planting. Fumigation is a method of pest control that fills an area w/ gaseous pesticides that suffocated/poison the pests within 2) Insecticides are added @ the time of planting 3) Herbicides are applied 2 times to get rid of weeds 4) Chemical fertilizers are applied 10 times 5) Fungicides are applied 8 times 6) Nerve toxins are applied 2 times to kills aphids

What 3 developments lead to the genetic engineering of plants? Explain and define.

1) Recombinant DNA technology and molecular cloning: Recombinant DNA technology combine two DNAs ofdifferent origins together. 2) A vector to transfer DNA of interest (Agrobacterium): For modification of important crop plants. And as a tool to understand plant genes and how they are regulated. 3) The ability to manipulate plant cells. Indeed, plant cells are totipotent= you can make an entire plant from a single cell.

If you were to design a "transgene" to transfer a gene of interest (GOI) to plant cells, what 4 different important DNAs would the transgene contain?

1. A promoter DNA element to allow transcription 2. Gene of Interest 3. Marker gene 4. T-DNA borders

There are 7 different biological steps that Agrobacterium uses when it transfers DNA to plant cells. Describe 4 of the steps that occur within the bacterial cell before the DNA is transferred to the plant.

1. Agrobacterium ​attracted​ ​to​ ​wounded​ ​cell​ ​because​ ​the​ ​cell​ ​produces​ ​a​ ​signal that ​turns​ ​on​ ​the​ ​ vir​ ​genes​ ​in​ ​the​ ​agrobacterium 2. VirD2 ​and​ ​VirE2​ ​attach​ ​to​ ​the​ ​T-DNA 3. VirD2​ ​produces the T-strand. VirE2​ ​attaches​ ​to​ ​the​ ​T-DNA​ ​borders​ ​and begins transcribing 4. T-complex ​is​ ​produced​ ​when​ ​VirD2​ ​attaches​ ​to​ ​5'​ ​end​ ​of​ ​T-strand​ ​and​ ​VirE2 covers ​the​ ​T-strand

What 2 factors were responsible for the "Green Revolution" in wheat?

1. Chemical fertilizers made plants grow faster and taller · However, there was no increase in grain yield 2. Norman Borlaug discovered a wheat variety that was semi-dwarf (defect of plant's ability to respond to GA) and had a high yield of grain · These dwarf plants could grow a lot under fertilizers

What are the basic steps involved in making an entire plant from a single cell?

1. Obtain protoplasts 2. Place them on a plate w/ the right hormones (auxin & cytokinin) 3. Formation of callus 4. Addition of cytokinin to form shoots, which will then form auxin to create roots 5. New Plant growth

Say you have 3 pieces of plant tissue. Place each on 3 different ratios of auxin to cytokinin. Make up some auxin to cytokinin ratios and then say what the plant tissue looks like when grown on these different ratios of auxin and cytokinin.

10:10 High auxin & cytokinin = callus (a mass of undifferentiated cells) 10:0 High auxin & Low cytokinin = large root growth 0:10 Low auxin & High cytokinin = large shoot and leaf growth

Give 2 unique features of ethylene compared to other hormones. Name 3 effects of ethylene during the life of a plant. Name 3 factors that stimulate the plant to make ethylene.

2 unique features of ethylene: Gas Can be produced internally and externally Made anywhere inside of a plant 3 effects of ethylene during the life of a plant: Change in color of a plant's fruit Ripening of fruit Senescence (aging) 3 factors that stimulate ethylene production: PWC Changes in the environment (i.e. flooding, drought, chilling) Wounding Pathogen attack

What is a clone? What is a bacterial colony? [If you transformed a single bacterial cell with a plasmid carrying a GOI, and the cell divides and divides over and over again, it will produce a "colony". The colony is a clone of cells, and the cells will have also "cloned" the plasmid with the GOI.] OK?

A clone is an organism or cell, or group of organisms or cells, produced asexually from one ancestor or stock, to which they are genetically identical. A bacterial colony is defined as a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike.

What are food subsidies? Pros? Cons?

A form of financial support given by the government → intended to provide stability to farmers and strengthen rural communities PROS: Farm subsidies support corn farmers Helped farmers a lot during the Great Depression when 45% of the US population lived on farms CONS: Few people get the money from subsidies and they are already rich Those that get subsidies practice industrial scale agriculture which is not environmentally friendly nor sustainable Hurts farmers that grow carrot and other healthy vegetables

Draw and explain the molecular bases for polar auxin transport in a vertical stack of 2 rectangular plant cells.

A polar (top to bottom) transport of auxin Auxin imported by influx carrier proteins, exported by efflux carrier proteins When auxin comes into the plant, it tends to get transported downwards and out because efflux proteins are located at the bottom of the cell CONSEQUENCE: APICAL DOMINANCE

Draw and explain how is the T-strand made?

Agrobacterium is attracted to wounded plant cells. Wounded plant cells produce small "signal" molecules that are taken up by Agrobacterium and act to turn-on (activate) the expression of the virulence (vir) genes. Once the vir genes are expressed, vir proteins (VirD2) are used to make a transferable copy of the T-DNA, called the "T-strand".

How does "antisense" technology work? Draw and explain the molecules are involved and what happens when they are present together in the cell?

Antisense ​technology​ ​works​ ​by​ ​pairing​ ​the​ ​targeted​ ​sense​ ​mRNA​ ​that​ ​produces​ ​the undesired ​protein​ ​with​ ​an​ ​antisense​ ​mRNA​ ​that​ ​possesses​ ​complementary​ ​bases.​ ​ When​ ​the two ​strands​ ​base​ ​pair,​ ​the​ ​mRNA​ ​cannot​ ​be​ ​translated​ ​into​ ​a​ ​protein. DNA → RNA anti-sense RNA and sense RNA, double-stranded RNA, complementary base pairing 2pts for explaining the outcome of antisense technology (examples include: no protein; no translation; ribosome can't translate; dsRNA degraded).

Define "apical dominance". What happens when the apex of a plant is removed? Why causes the plant to respond this way? Draw and use words.

Apical Dominance The auxin at the tip of the plant prevents lateral bud growth, which inhibits the formation of side shoots When the apex is removed → lateral shoots grow since auxin no longer inhibits growth cut off tip, lateral shoots grow

Recombinant DNA technology is based on 3 findings. One finding is bacterial plasmids. Describe bacterial plasmids and why they are important.

Bacterial plasmids are small circular DNA molecules found in bacteria They carry antibiotic resistance genes that can be transferred from one bacteria to another These genes are used extensively in molecular cloning and genetic transformation as they provide selectable marker genes that give advantage to cells containing these genes

What is bacterial transformation? How can you select for plasmid clones following transformation of bacteria with plasmid DNA? (I'm assuming it means pick out bacteria that transformed with desired DNA from those that didn't)

Bacterial transformation is the process where bacteria takes up DNA from the environment and then expresses its newly acquired genes. You can cut DNA into small fragments and will separate in gel electrophoresis the DNA can be added to similar plasmid DNA for cloning.

What are the two important parts of the CRISPR/Cas system. What does each of them do?

Cas9 Enzyme → Cuts the two strands of DNA @ a specific location in the genome so that bits of DNA can then be added or removed. gRNA → This consists of a small piece of pre-designed RNA sequence (about 20 bases long) located within a longer RNA scaffold. The scaffold part binds to DNA and the gRNA 'guides' Cas9 to the right part of the genome and the Cas9 enzyme cuts at the right part

Compare and contrast 3 differences between classical breeding and genetic engineering.

Classical breeding is slower Classical breeding is random and involves recombination throughout the entire genome and then selection for traits GE is precise as you insert a GOI into the genome Classical breeding can only be performed with very related species GE can take a gene from any organism and insert into another organism's genome.

Compare 3-4 issues of concern from Big Agriculture versus 3-4 sustainable practices in organic farming?

Concerns from Big Agriculture Huge farms needs lots of chemical inputs from fertilizers Big business monopolies run them and gets expensive Huge health issues (esp farm workers) Big agriculture can lead to lots of suicides Organic Farming Small scale and manageable Sustainable and renewable (friendly to the environment) More diverse species of a single crop are planted Crops are rotated so bugs don't get out of control when they like a particular crop and lay their eggs

Please describe in words the CRISPR "gene drive" system that is proposed to be used to wipe out mosquito populations.

Creating a transgenic insect that always will express the Cas9 enzyme and the guide RNA. forcing them to have the Cas9 and guide RNA We could design guide RNA to target a gene that will cause the mosquito to become sterile Cas9 and the guide RNA will then cut and destroy the gene Then the mosquitoes would reproduce and the next generation would be sterile (the process of gene drive eliminating mosquitoes)

Charles Darwin did a very simple experiment to show that there was "something" at the tip of the coleoptile (the protective sheath covering the emerging shoot of monocots) that caused it to bend towards light. Draw and describe this simple experiment.

Darwin OBSEVED that plants bend towards light Covered the tip of the plant with foil → no response Cover the rest of the plant with sand and leave the tip exposed → plant BENT towards the light Conclusion: SUGGESTED There must be something at the tip. Later discovered to be the hormone Auxin

What are embryonic stem cells versus adult stem cells? Compare their origin and their potential for development. Are there ethical problems in harvesting one type of stem cell versus the other?

Embryonic Stem Cells → cells with the ability to develop into any type of cell (pluripotent) Embryonic stem cells are extracted from embryos created by in vitro fertilization Adult Stem Cells → cells that an only morph into cell types within their tissue of origin ETHICAL PROBLEMS: There has been a large debate over the ethics of using embryonic stem cells. People debate whether or not it is humane to use potential cells (that could be life) to use for test subjects. Is it important to sacrifice some embryonic cells to benefit for the rest of human life? So far, only adult stem cells have been tested in humans. But now scientists are able to revert a skin cell back to its pluripotent state — like a stem cell — which raises fewer ethical concerns.

Why is industrial agriculture "fatal"? Name 3 causes of this fatality.

Environmentally, financially, socially (suicides), US farming practices are heavily reliant on chemical inputs such as fertilizers, laborers physically getting harmed while applying fertilizers

What is the major hormone to cause flower senescence? Why are orchids long-lived?

Ethylene causes flowers to senescence Usually, pollination → ethylene production in the petals → petals aging and dying However, in orchids pollination does not occur readily Since the anthers (which produce pollen) are not in easy reach of the female organ (stigma) Orchid flowers last long = little or no pollination

What are factors that lead countries to be wary of GMOs. Discuss Africa versus Europe versus the US.

European countries = anti-GMO Due to Mad Cow disease was transferable to humans via meat People lost faith in the ability of the government to make safety rules over food Africa and the 3rd world anti-GMO Most African countries ban GMO use for political reasons → they might lose aid from Europe and cannot export GM products to Europe It is also difficult to get neighboring countries to agree to plant GM crops Many trials of research needs to be done to prove that there are no negative effects before planting → national conflict United States We are still wary, but we have produced a lot already China wants our GMO corn

What are the evolutionary advantages to making fruits? What are the advantages of making fruits attractive to animals to eat?

Fruits have 2 benefits to plants: They protect plant seeds They facilitate the dispersal of the seed Animals are attracted to the fruits, eat them, and then poop out the seeds 3 Advantage of having the seeds pass through the digestive tracts of animals: Gastric stomach fluids break down the hard seed coat and release the embryo Animals will poop the seeds in other regions (seeds will be spread) Seed will come back in animal excrement, which is a highly nutritious form of compost for plant growth

What are "Round-up ready" seeds (in soybean)? What are advantages and disadvantages to Roundup transgenic plants?

GMO seeds that carry a gene that detoxifies Round-up, so plants can grow in the presence of Round-up weed killer Advantages: Farmers get good yields and it is easy to use this approach Glyphosate (inside of Round-Up) is better than other major herbicides (like Atrazine which disrupts endocrine hormone signaling in animals and humans) Disadvantages: Monsanto makes you pay twice (for seeds and round-up) Approach favors industrial agriculture of the same crop over vast areas, which destroys the land Also, farmers need to add a lot of chemical fertilizers

Compare the functions of abscisic acid versus gibberellic acid. Mention the different ways they act "antagonistically".

Gibberellic Acid (GA) Can rescue dwarf mutant plants by promoting growth Some plants require it to flower Some seeds need GA to germinate since it stimulates enzymes that provide nutrients for young seedlings Abscisic Acid (ABA) Prevents seeds from germinating When seeds get wet during germination, moisture activates enzymes that degrade ABA How they act antagonistically: GA: Promotes bud growth, promotes seed germination ABA: Inhibits bud growth, promotes seed dormancy Balance b/w the two determines growth pattern

Industrial agriculture uses herbicides and pesticides. Which chemical is more harmful to animals? What are 2 major herbicides, and what are their impacts on animals? Name 2 pesticides and what are their impacts on animals?

Herbicides: Glyphosate → prevents chloroplasts from making essential amino acids Damages proteins in the liver of mice Atrazine → disrupts endocrine (hormone) signaling in animals and humans Pesticides: Methyl bromide Found to cause ozone depletion and possesses nerve toxins Methyl iodide A known carcinogen and neurotoxin that causes late-term miscarriages Poressesses nerve toxins that can hurt animals

Define a hormone. Where is auxin made and where does it act? Where is cytokinin made and where does it act? How does the plant decide whether to make a root versus a shoot? [Think about concentration and/or ratios of hormone concentrations.]

Hormone → chemical signals that are produced in one part of an organism and function in another Auxin made @ the top of the plant functions/acts at the bottom of the plant to stimulate root formation Cytokinin made in the roots (& transported up) Acts at the top of the plant by promoting leaf and shoot growth when the concentration of auxin is higher than cytokinin roots grow when concentration of cytokinin is higher then shoots grow

Draw and describe the experiment used to demonstrate the direction of auxin movement...where a slice from the coleoptile was placed in two orientations of the agar block, and the auxin could flow out in only one orientation, and the agar with the auxin could be used in a bioassay for auxin etc

In a cut segment of a coleoptile, auxin will flow in one orientation From the Apical end to the Basal end (from A → B) If you place the segment on the agar cube w/ A on top and B on bottom, then auxin flows into the block Otherwise, it does not work This is the POLAR (TOP TO BOTTOM) transport of auxin The Auxin IAA is imported with influx carrier proteins and exported by enflux carrier proteins. The location of the efflux proteins at the bottom is what brings the polar auxin from top to bottom of the plant.

What is thought to be the biological "rationale" for why Agrobacterium goes to all the trouble of transferring the T-DNA to plant cells?

In nature, Agrobacterium likely goes to all the trouble of transferring DNA to plants so that the tumors will provide a food source, called opines (used by the bacterium as an important source of nitrogen and energy) that are encoded by the T-DNA once inside the plant cell.

How do auxin and light cause bending? Light shining asymmetrically on one side will stimulate auxin production on the opposite side....and then what does auxin do to the cells....do they expand? How? Please draw and use words too.

Light stimulates auxin production on the opposite side away from the light source, so there is more auxin on the darker side of the coleoptile More expansion (due to water uptake) of the cells on one side versus the other side Bending is due to asymmetric cell expansion in response to auxin and light

Trouble with neonicotinoids"! Treating seeds with neonicotinoid-type insecticides immediately kills insects on the surface of the plants. Name 4 other effects of this insecticide that affect the plant itself or non-targeted organisms in the environment near the plant. Draw and use words.

Long term low doses cause bee hives to collapse worker bees to lose their sense of smell/memory/locomotion/eat less. also cause they to have immune deficiency The queen bee is the most affected, and they are unable to reproduce as effectively to replenish the lowering numbers of worker bees in the colonies. Eventually, the biomagnification affects predators

How was Dolly the sheep cloned? Is there a conceptual link between the initial steps of making dedifferentiated sheep cells and dedifferentiated plant cells in callus culture?

Mammary cells were put into a culture with low nutrients and became de-differentiated so they no longer expressed the genes to be a mammary cell The nucleus from these cells was placed into an enucleated egg cell Electric shocks were used to stimulate fusion between the donor and egg cells → stimulated cell division The egg cytoplasm contained signals and information on how to make an embryo → was able to self develop from there CONCEPTUAL LINK: Both take adult cells and dedifferentiated them by using external stimuli (totipotent plant cell and dedifferentiated animal cells) Sheep cells: Starve the adults cells and cause them to dedifferentiate Adult leaf cells: Supply auxin and cytokinin to induce callus growth Then give the right hormones so that the organism can form from that single cell

Chlorpyrifos is a nasty pesticide. Why? Dow produces it and the EPA has not yet banned it. Do you think it will become banned during the current political climate?

Moderately toxic to humans → neurological effects, persistent developmental disorders, and autoimmune disorders I do not think that it will be banned → Trump is focused on reversing the legacies of the Obama administration (including environmental regulations) Trump cut down the EPA staff and its power by dismissing environmental regulations → it will be hard for the EPA to pass more regulations under this administration → Trump just cares about catering to Big business and had his campaign funded by Dow Chemicals

What factors that led to the dramatic 90 % loss of monarch butterfly populations?

Monarch butterflies like to eat milkweed plants, not BT corn Greater use of the herbicide Roundup has coincided with a sharp decline in the amount of milkweeds Monarch butterfly reproduction experiencing heavily declines Weed control is driving the eradication of the milkweed plant that the monarch exclusively lays its eggs on

What is Monsanto's new herbicide-transgenic plant combo? What are issues of concern regarding this strategy?

Monsanto gets you to pay twice, you need to buy the seeds and the Round-Up which favors industrial agriculture monocultures (of the same crop) which destroys the land after adding tons of chemical fertilizers. Roundup Ready crops are crops genetically modified to be resistant to the herbicide Roundup. Roundup is the brand-name of a herbicide produced by Monsanto. Its active ingredient glyphosate was patented in the 1970s. Roundup is widely used by both people in their backyards and farmers in their fields. Roundup Ready plants are resistant to Roundup, so farmers that plant these seeds must use Roundup to keep other weeds from growing in their fields. These crops were developed to help farmers control weeds. Because the new crops are resistant to Roundup, the herbicide can be used in the fields to eliminate unwanted foliage. Current Roundup Ready crops include soy, corn, canola, alfalfa, cotton, and sorghum, with wheat under development. Roundup Ready crop seeds have notoriously been referred to as "terminator seeds." This is because the crops produced from Roundup Ready seeds are sterile. Each year, farmers must purchase the most recent strain of seed from Monsanto. This means that farmers cannot reuse their best seed. Read more about terminator seeds.

What are the general pros and cons of making GMO plants that express herbicides or pesticides

PROS: Farmers are using less pesticides (even in the industrial world) GMO tech shortens the time to make disease resistant plants Seeds designed to resist pests are more advantageous to small farmers in the 3rd world CONS: Biotech only favors big business May lead to the evolution of super weeds Biotech companies have a monopoly GMO plants will cause the development of resistant pests

Design your own restriction enzyme (RE) site with 6 palindromic nucleotides. Make the site be recognized by an enzyme that cuts asymmetrically to produce "sticky" ends. Draw this restriction site on a plasmid cut with the RE and on the ends of a piece of DNA cut with the same RE. Show how the plasmid sticky ends can "recombine" with the sticky ends of the DNA cut with the same RE.

Palindrome in DNA has a different literary context compared to the English meaning (racecar). In DNA, the palindrome takes both strands of DNA into consideration. The plasmid sticky ends can "recombine" with the sticky ends of the DNA through DNA ligase (check study guide for diagram)

What are 4 advantages of making vaccines in plants?

Plants grow fast and are inexpensive to grow Plant material can be easily harvested to produce vaccines Plants can grow entire fields and produce lots of useful vaccine With plants, we avoid harming animals

What is the major difference between reproductive cloning versus therapeutic cloning?What are the major ethical differences between them?

Reproductive Cloning (i.e. Dolly the Sheep) → making an entire individual capable of reproduction ETHICS: You could unintentionally create a defective person/animal that needs extensive care or might die early Therapeutic Cloning → (somatic cell nuclear transfer) make stem cells capable of producing different cells and tissues that can be used to replace non-functional tissues in animals/humans ETHICS: Only cells and tissues (somatic) are manipulated

Define restriction enzymes, sticky ends, and palindromes. How do restriction enzymes facilitate molecular cloning strategies with plasmids?

Restriction enzymes- proteins that cut DNA at specific sequences Sticky ends- unpaired nucleotides produced by the asymmetric cutting of the DNA Palindromes- sequence that reads the same, backwards as forwards, ON THE COMPLIMENTARY STRANDS All DNAs cut with a particular restriction enzyme will have the same sticky ends that are complementary to each other! Cut plasmid and GOI with SAME enzyme in order to join the complementary ends together. DNAs can recombine with each other by the complementary base pairing mediated by their sticky ends.

What is a selectable marker gene? What is a non-selectable marker gene? Gives examples of each.

Selectable Marker Gene → produces a phenotype that gives a selective advantage to the cells that carry the gene Non-selectable markers provide a visual trait that allow identification of cells that have taken up GOI (e.g. GFP, GUS, pigmentation). The advantage of a selectable marker is that you can produce a pure population of mutants, reducing the need to screen them individually. Non¬selectable markers allow the growth of untransformed cells, which is useful to compare as a control, if necessary.

Rice is a very important crop. How did scientists engineer rice crops to be flood tolerant? [In this case they took a gene from a flood tolerant crop and put this gene into a flood sensitive crop.] Thus, they engineered rice with a rice gene; is this more natural? What are the advantages of using this method? Why is flood tolerance in rice so important? (Flood tolerant rice was described briefly in lecture ...if you are interested in more info watch FYI TED talk by Pam Ronald).

Studied an ancient variety of rice that had an amazing property to withstand two weeks of complete submergence (Pam Ronald) Breeders developed a rice variety carrying the Sub1 gene using precision breeding This is technically more natural because it is less invasive and less adverse effects (ex. strawberries) Sub1 gene allows for rice varieties to survive being submerged under water for more than 3 days Scientists at UC Davis identified the FR13A flood resistant trait in Indian rice varieties and added it to a high yielding rice variety via genetic engineering

What is sustainable agriculture? Name 4 production practices that are essential for such good farming to occur.

Sustainable agriculture refers to agricultural production that can be maintained without harming the environment Has 3 goals: environmental stewardship, farm profitability, and prosperous farming communities 4 Production Practices: Reduced/no use of chemical fertilizers (to prevent runoff) Reduced/no use of chemical pesticides (avoid killing beneficial insects) Plant a variety of crops (no monocultures) Use of organic fertilizers (chicken or cow manure, compost, etc.)

Agrobacterium: What is the T-DNA? What is the T-strand? What are T-DNA borders? What are the virulence genes? How does Agrobacterium cause "crown gall tumors" to form?

T-DNA = DNA from the tumor-inducing plasmid. T-strand = transferrable copy of the T-DNA T-DNA borders: 25 nucleotide base pairs at the ends of T-DNA. Any DNA in between the borders will be transferred from the Agrobacterium to the plant cell. The virulence genes allow the bacteria to transfer its DNA Agrobacterium causes the plant to produce high concentrations of cytokinin and auxin, which creates crown gall tumors

What is a bioassay? Give an example.

Testing a biological system for a response to a chemical substance @ low concentrations, low responses @ high concentration, more response Graph looks like a logistic curve w/ a carrying capacity I.e. Amount of bending and concentration of Auxin

Use the "book" metaphor to explain how genetic engineering is more precise than classical breeding for traits.

The DNA is like a big ass book containing hundreds of millions of pages of instructions for all things life. Classical breeding is like replacing a couple of chapters of this book with chapters from another book and hoping that the instructions are vaguely what you want. Genetic engineering however is much more accurate, targeting specific DNA sequences, which is akin to switching out specific words and sentences in this big book.

What is the T-complex? What is special about the proteins in the T-complex? How do they function in the plant cell?

The T‐complex is the protein/DNA complex that contains the T‐strand and proteins VirD2 and VirE2 and travels from the agrobacterium into the plant cell. These proteins have a sequence of amino acids on them that is recognized in the plant cell as a nuclear localization signal. These are special because while bacteria have no nucleus and they have no need of this signal in their cell, eukaryotes with multiple compartments in the cell use sequence tags like this to move proteins to specific compartments where that protein functions. The proteins function in the plant cell by protecting the T‐strand from plant enzymes that could degrade it, and by having this NLS signal on them. In the plant cell, plant proteins recognize this sequence and direct the T‐complex to the nuclear pore and into the nucleus, where the T‐strand can then insert into the host genome

When the Cas enzyme cuts both strands of the DNA, this will lead to small alterations in the genetic code that can cause 3 possible effects. What are they?

The coded amino acid changes Altered reading frames that will lead to an early stop codon → protein is shorter than normal and defective A stop codon at the site of repair

What is the "dead" zone? How is it caused? Explain.

The dead zone is in the Gulf of Mexico, absent of marine life. The fertilizer run off causes huge algal blooms and when the algae die, they sink to the bottom of the ocean. This dead algae becomes decomposed by bacteria, which use up a lot of oxygen in the process. The lack of oxygen causes marine animals to die.

Draw a sunflower at sunrise or sunset. Where are the flower heads in relation to the sun? Where is the auxin concentration highest in the stems of the flowers? What does this high auxin cause?

The flower heads follow the direction of the Sun. The auxin concentration is highest in the stems of the flowers on the shaded side of the stem in order to cause the curvature that we can see. As the Sun passes throughout the day, the auxin is produced on the other side as it begins to become the shaded region of the stem.

How is it that auxin and cytokinin cause such dramatic effects on overall plant growth? Discuss how their presence in cells leads to the expression (transcription and translation) of whole sets of genes. How can this be easily controlled (clue = what do you need to start transcription?)?

Transcription requires RNA polymerase & transcription factors Hormones (auxin) can activate transcription factors → these factors bind to specific promoters → stimulates transcription of genes During gene expression → small amounts of hormones signal protein CASCADEs, which amplify the hormone signal and lead to greater production of proteins.

What are 4 advantages of the CRISPR/Cas system?

Very precise and will modify a gene of interest in the genome No other sites are affected Generates heritable changes in a single generation Can "knock out" unwanted genes (like cancer or sickness causing genes) or can "knock in" useful genes/traits Can be applied to any biological system or organism (not specific to one)

Fritz Went "proved" there was something at the tip. What did he do? Draw and explain with words.

Went PROVED (Darwin SUGGESTED) that there is a growth promoting substance at the plant tip Cut off the tip of a coleoptile Placed it on an agar cube and allowed the chemicals within the tip to diffuse into the cube He then decided to place the agar cube w/ the infused chemicals onto the top of the cut coleoptile in 2 ways: Either in the middle → coleoptile grew upwards and straights Asymmetrically → coleoptile grew in a curved direction He concluded that there must be something within the original tip that went into the agar and caused bending & growth for the plant in response light

Explain the natural defense system in plant or animal cells, called RNA interference, in simple words. For example, what happens in a cell that is making too much of an RNA (for example, many viruses are RNAs) or an mRNA (like too much mRNA for the purple color in the petunias that turned white)?

When there is an abundance of a particular RNA, such during infection with plant viruses (which make a huge number of RNA copies of their genomes) or during genetic engineering experiments where novel genes are added to the plant genome, the plant cell tries to get rid of this extra RNA. Thus the cell gets rid of the extra overly abundant RNA by making interfering RNA that is complementary to the overly abundant RNA, and this leads to the inability of the over abundant RNA to be translated......and there are also special enzymes in the cell that destroy double stranded RNA. RNA silencing is like an fight-back defense response in plants,analogous to the immune response in animal cells.

Why is it important to have a both a "marker" gene and a gene of interest in the construct that you design for genetic engineering of plants?

Without a marker gene, it can be hard to know what cells in the plant were actually affected by the GOI. Marker genes produce a phenotype so that you can know that your GOI was transferred to the plant cell This is because the two transgenes are directly adjacent to one another in the transgene Both are within the T-DNA borders

So now you know how to clone different DNAs into a plasmid. Draw and use words for what pieces of DNA you would "clone" into a small plasmid (that already has T-DNA borders) to express a gene of interest (GOI) and a selectable marker gene. Make sure you show sticky ends of DNA in the fragments you are cloning. (This plasmid later could be put into Agrobacterium and it would be transferred to the plant cell.)

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So now you know how to clone. Say you want to test whether a gene of interest (GOI) makes a protein that carries nuclear localization signal sequences (or any other type of cellular localization sequence). Draw and use words to create (by recombinant DNA technology) a gene that will encode a "fusion" protein. What type of gene would you clone to be fused with the GOI? Mention in words what is important to consider at the "fusion" site?

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